It is often necessary to obtain quantitative information that characterizes the features of a surface layer of microscopic objects relatively free from the influence of underlying structures. Light-scattering spectroscopy (LSS) is one technique that can provide the desired information. One such application is monitoring the precancerous condition of the cells in the epithelial layer that cover surfaces or body organs.
The ability to measure quantitative changes in intracellular structures in situ provides an opportunity for early diagnosis of cancer or precancerous lesions. More than 90% of all cancers are epithelial in origin. Most epithelial cancers have a well-defined precancerous stage characterized by nuclear atypia and dysplasia. Lesions detected at this stage can potentially be eradicated with early diagnosis. However, many forms of atypia and dysplasia are flat and not visually observable. Thus, surveillance for invisible dysplasia employs random biopsy, followed by microscopic examination of the biopsy material by a pathologist. However, usually only a small fraction of the epithelial surface at risk for dysplasia can be sampled in this way, potentially resulting in a high sampling error.
Body surfaces are covered with a thin layer of epithelial tissue. The thickness of the epithelium in various organs ranges from less than 10 μm in simple squamous epithelia (having single layer of epithelial cells) to several hundred μm in stratified epithelia that have multiple layers of epithelial cells. Beneath all epithelia are variable layers of the supporting components including relatively hypocellular connective tissues, inflammatory cells, and neurovascular structures.
For example, in hollow organs, such as the gastrointestinal tract, the epithelial cell layer is from 20 μm to 300 μm thick, depending on the part of the tract. Below the epithelium is a relatively acellular and highly vascular loose connective tissue, the lamina propria, that can be up to 500 μm in thickness containing a network of collagen and elastic fibers, and a variety of white blood cell types. Beneath the lamina propria there is a muscular layer, the muscularis mucosae, (up to 400 μm thick) and below that, another layer, about 400-600 μm thick, of moderately dense connective tissue containing many small blood vessels and abundant collagen and elastic fibers called the submucosa. The overall thickness of those layers is about 1 mm. Since a characteristic penetration depth of optical radiation into biological tissue does not usually exceed 1 mm, for a preferred embodiment it is sufficient to limit measurements of tissue by those layers.
Light traversing the epithelial layer can be scattered by cell organelles of various sizes, such as mitochondria and nuclei, which have refractive indices higher than that of the surrounding cytoplasm. Elastic scattering of light by cells is due to a variety of intracellular organelles, including mitochondria, a variety of endosomes and other cytoplasmic vesicles, nucleoli, and nuclei. The smaller organelles are responsible for large angle scattering, whereas the nucleus contributes to scattering at small angles.
The cell nuclei are appreciably larger than the optical wavelength (typically 5-10 μm compared to 0.5 μm). They predominantly scatter light in the forward direction, and there is appreciable scattering in the backward direction, as well. The backscattered light has a wavelength-dependent oscillatory component. The periodicity of this component increases with nuclear size, and its amplitude is related to cellularity or the population density of the epithelial nuclei. By analyzing the frequency and amplitude of this oscillatory component, the size distribution and density of epithelial nuclei can be extracted.
In order to detect changes in epithelial cell nuclei associated with dysplasia, the light reflected from the epithelial layer must be distinguished from the light reflected from the underlying tissue. Since, as noted above, the penetration depth in tissue substantially exceeds the epithelial thickness, the backscattered light from epithelial nuclei is ordinarily very small in amplitude, and it is easily masked by the diffuse background of light reflected from the underlying tissue. This diffuse background reflected light must be removed in order to analyze the backscattered component.
Previous approaches have sought to remove the diffuse background reflected light by modeling the general spectral features of the background. However, this approach must be specifically adapted to each different type of tissue studied, and its accuracy is theory dependent. More robust methods of removing or significantly reducing the diffuse component of the scattered light are needed to extend the use of LSS to various medical applications.
The various forms of epithelial dysplasia exhibit some common morphological changes on microscopic examination, the most prominent of which relate to the nuclear morphology. The nuclei become enlarged, pleomorphic (irregular in contour and size distribution), “crowded” (they occupy more of the tissue volume), and hyperchromatic (they stain more intensely with nuclear stains). The diameter of non-dysplastic cell nuclei is typically 5-10 μm, whereas dysplastic nuclei can be as large as 20 μm across.
Epithelial cell nuclei can be modeled as transparent spheroids that are large in comparison to the wavelength of visible light (0.4-0.8 μm), and whose refractive index is higher than that of the surrounding cytoplasm because of their chromatin content. The spectrum of light backscattered by these particles contains a component that varies characteristically with wavelength, with this variation depending on particle size and refractive index.
The light scattering from epithelial cell nuclei can be isolated using polarized light. It is known that polarized light loses its polarization when traversing a turbid medium such as biological tissue. In contrast, polarized light scattered backward after a single scattering event does not lose its polarization. This property of polarized light has been used previously to image surface and near surface biological tissues. Thus, by subtracting the unpolarized spectral component of the scattered light, the portion of light scattering due to backscattering from epithelial cell nuclei can be readily distinguished. The difference spectrum can be further analyzed to extract the size distribution of the nuclei, their population density, and their refractive index relative to the surrounding medium.
Although many epithelial cancers are treatable provided they are diagnosed in a pre-invasive state, early lesions are often almost impossible to detect. Before they become invasive, at stages known as dysplasia and carcinoma in situ, early cancer cells alter the epithelial cell architecture. In particular, the nuclei become enlarged, crowded and hyperchromatic, that is, they stain abnormally dark with a contrast dye. These pre-invasive signs have been detectable by histological examination of biopsy specimens, but no reliable optical technique to diagnose dysplasia in-vivo is available. Light-scattering spectroscopy (LSS) can provide a biopsy-free means to measure the size distribution and chromatin content of epithelial-cell nuclei as an indictor of pre-invasive neoplasia.
For a collection of nuclei of different sizes, the light-scattering signal is a superposition of these variations, enabling the nuclear-size distribution and refractive index to be determined from the spectrum of light backscattered from the nuclei. Once the nuclear-size distribution and refractive index are known, quantitative measures of nuclear enlargement, crowding and hyperchromasia can be obtained.
Barrett's esophagus is a pre-cancerous condition arising in approximately 10-20% of patients with chronic reflux of stomach contents into the esophagus. People who develop Barrett's esophagus may have symptoms of heartburn, indigestion, difficulty swallowing solid foods, or nocturnal regurgitation that awakens them from sleep. Patients with Barrett's esophagus have an increased risk of developing esophageal adenocarcinoma, the most rapidly increasing cancer in the United States.
Adenocarcinoma of the esophagus arises in metaplastic columnar epithelial cells in the esophagus, as a complication of such chronic gastrointestinal reflux. In this condition, the distal squamous epithelium is replaced by columnar epithelium consisting of a one cell layer which resembles that found in the intestines. Barrett's esophagus is frequently associated with dysplasia, which later can progress to cancer. Trials of endoscopic surveillance of patients with Barrett's esophagus have not resulted in a reduction of esophageal cancer mortality. The most likely explanation is that dysplasia occurring in the esophagus cannot be seen with standard endoscopic imaging and sporadic biopsy sampling is necessary. This procedure can sample only about 0.3% of the tissue at risk. Thus, there is tremendous potential for sampling error.
The application of optical techniques to diagnose dysplasia in Barrett's esophagus is limited by the fact that the primary alterations in the tissue occur in the epithelium which is one cell thick (˜20-30 μm) while fluorescence or reflectance spectra are mostly formed in deeper tissue layers. One of the most prominent features of a dysplastic epithelium is the presence of enlarged, hyperchromatic, and crowded nuclei. In fact, these changes in nuclear size and spatial distribution are the main markers used by a pathologist to diagnose a tissue specimen as being dysplastic. No significant changes are observed in other tissue layers. Unfortunately, epithelium does not contain strong absorbers or fluorophores, and the thickness of the epithelium is relatively small and thus negligible. These make epithelium diagnosis in Barrett's esophagus a difficult problem. In such cases, LSS can provide a biopsy-free means to measure the size distribution and chromatin content of epithelial-cell nuclei as an indictor of pre-invasive neoplasia.